Confidence evaluation of the targeted LC-MS/MS and DDA-LC-MS/MS phosphoproteome investigation. Using the MD-score and PhosphoRS methods, we showed the improvement of the targeted LC-MS/MS-based phosphoproteome investigation over DDA-based analysis. (a) The comparison of the assessment results by Mascot Delta Score (MD-score). The scores of the identified pTyr peptides in the targeted LC-MS/MS runs are higher than those identified pTyr peptides in DDA LC-MS/MS runs (19.24 ± 0.19 vs. 17.55 ± 0.68). (b) The comparison of the assessment results by the phosphoRS algorithm in Proteome Discover Software. 83 pTyr peptides (83/191) identified in the targeted LC-MS/MS runs pass the validation. Further, 66 of these validated phosphorylation localization are of high confidence (with probability higher than 0.8). In comparison, only 55 pTyr peptides (55/189) identified in the pure DDA LC-MS/MS runs do. And only 43 of them are of high confidence.